Compositions and methods for detection of drug resistant Mycobacterium tuberculosis

ABSTRACT

Methods for the rapid detection of the presence or absence of  Mycobacterium tuberculosis  (MTB) resistant to rifampicin (MTB-RIF) and/or MTB resistant to isoniazid (MTB-INH) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the genes for rpoB, inhA, and katG, along with kits are provided that are designed for the detection of MTB-RIF and/or MTB-INH.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 17, 2014, is named32319-US_SL.txt and is 138,013 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of virus diagnostic, and moreparticularly, to PCR detection methods utilizing hydrolysis probes fordetection of drug resistant Mycobacterium tuberculosis.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a bacterial disease caused by various strains ofmycobacteria, such as Mycobacterium tuberculosis (MTB) most often foundin the lungs. It is transmitted from person to person through the airwhen individuals with pulmonary or laryngeal tuberculosis, cough,sneeze, or spit, and propel MTB into the air. It is estimated thatone-third of the world population is infected with MTB and 9 millionpeople develop TB each year. TB continues to be a leading cause of humaninfectious disease and drug-resistant strains of MTB are on the rise,especially in developing countries.

Two common first-line drugs for the treatment of MTB include isoniazid(INH) and rifampicin (RIF), and patients can acquire drug resistant MTBfrom living in or visiting a place where drug resistance is prevalent.Patients can also develop drug resistant MTB when their antibiotictreatment regimen is interrupted. Culturing on solid or liquid media isstill considered the gold standard for MTB and MTB drug resistancedetection, but culturing can take up to eight weeks for results. Manycommercial nucleic acid tests for MTB drug resistance have a very fastturn-around time, but cannot detect a population with a small percentageof mutant species in a mixed infection containing both wild type andmutant species. Thus there is a need in the art for a quick and reliablemethod to specifically detect MTB resistant to rifampicin (MTB-RIF)and/or MTB resistant to isoniazid (MTB-INH) in a sensitive manner.

SUMMARY OF THE INVENTION

Embodiments described herein relate to methods for the rapid detectionof the presence or absence of MTB-RIF and/or MTB-INH in a biological ornon-biological sample, for example, multiplex detection of MTB-RIFand/or MTB-INH by real-time polymerase chain reaction in a single testtube. Embodiments include methods of detection of MTB-RIF and/or MTB-INHcomprising performing at least one cycling step, which may include anamplifying step and a hybridizing step. Furthermore, embodiments includeprimers, probes, and kits that are designed for the detection of singleMTB-RIF or MTB-INH, or MTB-RIF and MTB-INH co-infections in a singletube. The detection methods are designed to specifically identify singlepolymorphism (SNP) in target MTB genes for rpoB (beta subunitprokaryotic RNA polymerase), inhA (enoyl-acyl carrier proteinreductase), and katG (catalase-peroxidase) simultaneously, which allowsdetection and differentiation of MTB-RIF and/or MTB-INH infections in asinglet test. For example, there are 17 SNPs in the rboB gene whichconfer resistance to rifampicin in MTB which include rpoB 531L, rpoB531W, rpoB 526L, rpoB 526Y, rpoB 526D, rpoB 526N, rpoB 513L, rpoB 513K,rpoB 513P, rpoB 522L, rpoB 522Q, rpoB 522W, rpoB 516V, rpoB 516Y, rpoB533P, rpoB 511P, and rpoB 526R; there are 3 SNPs in the inhA gene whichconfer resistance to isoniazid in MTB which include inhA-15T, inhA-8A,and inhA-8C; and there are 4 SNPs in the katG gene which also conferresistance to isoniazid in MTB which include katG 315I, katG 315N, katG315T, and katG 315T2.

In one embodiment, a method of detecting MTB-RIF and/or MTB-INH in asample is provided, including performing an amplifying step comprisingcontacting the sample with at least a set of rpoB primers, a set of inhAprimers, and a set of katG primers to produce one or more amplificationproducts if any rpoB, inhA, and katG target nucleic acid is present inthe sample; performing a hybridizing step comprising contacting said oneor more amplification products with a plurality of detectable rpoBprobes, a plurality of detectable inhA probes, and a plurality ofdetectable katG probes, including: 17 rpoB probes for detection of oneor more of 17 single nucleotide polymorphisms SNPs which conferrifampicin resistance to MTB; 3 inhA probes for detection of one or moreof 3 SNPs which confer isoniazid resistance to MTB; and 4 katG probesfor detection of one or more of 4 SNPs which confer isoniazid resistanceto MTB; and detecting the presence or absence of said one or moreamplification products, wherein the presence of said one or moreamplification products is indicative of the presence of MTB-RIF and/orMTB-INH in the sample and wherein the absence of said one or moreamplification products is indicative of the absence of MTB-RIF and/orMTB-INH in the sample; wherein said plurality of rpoB probes comprisehydrolysis probes for detection of each of the 17 SNPs which conferrifampicin resistance to MTB, comprising rpoB 531L, rpoB 531W, rpoB526L, rpoB 526Y, rpoB 526D, rpoB 526N, rpoB 513L, rpoB 513K, rpoB 513P,rpoB 522L, rpoB 522Q, rpoB 522W, rpoB 516V, rpoB 516Y, rpoB 533P, rpoB511P, and rpoB 526R; wherein said plurality of inhA probes comprisehydrolysis probes for detection of each of the 3 SNPs which conferisoniazid resistance to MTB, comprising inhA-15T, inhA-8A, and inhA-8C;and wherein said plurality of katG probes comprise hydrolysis probes fordetection of each of the 4 SNPs which confer isoniazid resistance toMTB, comprising katG 315I, katG 315N, katG 315T, and katG 315T2.

Another embodiment provides an oligonucleotide comprising or consistingof a sequence of nucleotides selected from SEQ ID NOs: 1 through 409, ora complement thereof, which oligonucleotide has 100 or fewernucleotides. In another aspect, the present disclosure provides anoligonucleotide that includes a nucleic acid having at least 70%sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) toone of SEQ ID NOs: 1 through 409, or a complement thereof, whicholigonucleotide has 100 or fewer nucleotides. Generally, theseoligonucleotides may be primer nucleic acids, probe nucleic adds, or thelike in these embodiments. In certain of these embodiments, theoligonucleotides have 40 or fewer nucleotides (e.g. 35 or fewernucleotides, 30 or fewer nucleotides, etc.) In some embodiments, theoligonucleotides comprise at least one modified nucleotide, e.g. toalter nucleic acid hybridization stability relative to unmodifiednucleotides. Optionally, the oligonucleotides comprise at least onelabel and/or at least one quencher moiety. In some embodiments, theoligonucleotides include at least one conservatively modified variation.“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids, which encode identical or essentially identical aminoacid sequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than 5%, more typically less than 4%, 2% or 1%) inan encoded sequence are “conservatively modified variations” where thealterations result in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ nuclease activity. Thus, the first and second fluorescent moietiesmay be within no more than 8 nucleotides of each other along the lengthof the probe.

In a further embodiment, a kit for detecting one or more nucleic acidsof MTB-RIF and/or MTB-INH is provided. The kit can include a pluralityof sets of rpoB, inhA, and katG primers specific for amplification of arpoB, inhA, and katG gene targets; and a plurality of detectable rpoB,inhA, and katG probes specific for detection of a rpoB, inhA, and katGamplification products.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor fluorescent moieties, or can include fluorophoricmoieties for labeling the probes. The kit can also include nucleosidetriphosphates, nucleic acid polymerase, and buffers necessary for thefunction of the nucleic acid polymerase. The kit can also include apackage insert and instructions for using the primers, probes, andfluorophoric moieties to detect the presence or absence of MTB-RIFand/or MTB-INH in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the drawings and detailed description,and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show wild type and mutant amplicon sequences for therpoB gene target and indication each of the 17 SNPs which conferrifampicin resistance to MTB.

FIGS. 2A and 2B show wild type and mutant amplicon sequences for theinhA gene target and indication each of the 3 SNPs which conferisoniazid resistance to MTB.

FIGS. 3A and 3B show wild type and mutant amplicon sequences for thekatG gene target and indication each of the 4 SNPs which conferisoniazid resistance to MTB.

FIGS. 4 and 4B show growth curve assay for singleplex PCR SNP-specificprobe detection using unmodified probe for 522L SNP compared to usingmodified probe for 522L SNP (MT target (˜1 e6, 1 e2, 1 e3 10c/PCR)compared with WT).

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of MTB-RIF and/or MTB-INH infections by nucleic acidamplification provides a method for rapidly and accurately detectingMTB-RIF and/or MTB-INH infections. A real-time assay for detectingMTB-RIF and/or MTB-INH in a sample is described herein. Primers andprobes for detecting the rpoB, inhA, and katG target nucleic acids ofMTB-RIF and/or MTB-INH are provided, as are articles of manufacture orkits containing such primers and probes. The increased sensitivity ofreal-time PCR for detection of MTB-RIF and/or MTB-INH compared to othermethods, as well as the improved features of real-time PCR includingsample containment and real-time detection of the amplified product,make feasible the implementation of this technology for routinediagnosis of MTB-RIF and/or MTB-INH infections in the clinicallaboratory.

Identification of drug resistant MTB requires detection of numeroussingle nucleotide polymorphisms (SNPs) in the MTB genome located onseveral different genes. Using a novel variation of hydrolysis probe(also known as TaqMan probe) design, a multiplex of highlydiscriminating TaqMan probes were created, wherein each TaqMan probe candetect a single SNP without cross reactivity. The probes are designed tobe very short and highly stabilized in order to bind and cleave withgreat specificity only to a perfectly matched drug resistant (mutant)sequence.

The present disclosure provides Taqman probes for detection of thevarious SNPs which confer resistance to MTB-RIF and MTB-INH. TaqMancompatible probes are not generally able to detect single base pairmismatches. Generally, TaqMan probes are designed to be much longer, andhave a significantly higher melting temperature than the associated PCRprimers in order to ensure adequate probe binding to the target sequenceprior to the primers and attain maximum probe cleavage during PCR. Dueto the high melting temperature and length of such probes, they aregenerally very tolerant of a single base mismatch under the probe regionand thus do not discriminate between two targets that differ by only asingle base. In the present disclosures, the hydrolysis TaqMan probesare able to successfully detect only drug resistant MTB and not crossreact with drug sensitive MTB, which can only differ by a single base.The present disclosure provides numerous short, highly modified probesthat can detect a single base pair mismatch under the probe region. Themodified bases that can be substituted in the probe designs includepropynyl-dC, t-butyl-benzyl dC, propynyl-dU, G-clamps, methyl-dC,N6-methl dA, and 7-deaza-dG. In designing the probes, the presentinventors strategically placed several modified base pairs within theprobe sequences in order to maximize the discrimination ability of theprobe. It was discovered that some modifications work better thanothers, and that the discrimination ability is affected by the placementof the modifications in the probe.

The present disclosure provide methods and kits for multiplexed assayswhich can utilize a plurality of highly modified TaqMan probes whereineach probe can detect a single SNP known to confer Rrifampicin (RIF) andIsoniazid (INH) drug resistance in the MTB genome without significantlycross reacting with drug sensitive (wild type) MTB. The unique abilityof the disclosed TaqMan probes to detect mutant SNPs without significantWT cross reactivity enables the assay to detect a minor presence of drugresistant MTB when mixed in a background of wild type (WT). The presenceof mixed infection has been reported, and it has been suggested that theprevalence of mixed infection is underreported due to the inability ofcurrent commercial assays to detect drug resistant MTB in the presenceof drug sensitive MTB. It is reported by the Center for Disease Control(CDC) that a patient that is infected with as little as 1% drugresistant MTB in a background of WT may fail their proposed treatmentregimen.

The methods may include performing at least one cycling step thatincludes amplifying one or more portions of rpoB, inhA, and katG nucleicacid molecule gene targets from a sample using a plurality of pairs ofprimers, including rpoB, inhA, and katG specific primers as used hereinrefer to oligonucleotide primers that specifically anneal to nucleicacid sequences encoding rpoB, inhA, and katG, respectively, and initiatesynthesis therefrom under appropriate conditions. Each of the discussedrpoB, inhA, and katG primers anneals to a target within or adjacent tothe respective rpoB, inhA, and katG target nucleic acid molecule suchthat at least a portion of each amplification product contains nucleicacid sequence corresponding to respective target. The one or more ofrpoB, inhA, and katG amplification products are produced provided thatone or more of rpoB, inhA, and katG nucleic acid is present in thesample, thus the presence of the one or more of rpoB, inhA, and katGamplification products is indicative of the presence of rpoB, inhA, andkatG in the sample. The amplification product should contain the nucleicacid sequences that are complementary to one or more detectable probesfor detection of the SNPs in rpoB, inhA, and katG which conferrifampicin and/or isoniazid resistance to MTB. Each cycling stepincludes an amplification step, a hybridization step, and a detectionstep, in which the sample is contacted with the one or more detectableprobes for rpoB, inhA, and katG for detection of the presence or absenceof MTB-RIF and/or MTB-INH in the sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., rpoB, inhA, andkatG nucleic acid molecules). Amplifying a nucleic acid moleculetypically includes denaturing the template nucleic acid, annealingprimers to the template nucleic acid at a temperature that is below themelting temperatures of the primers, and enzymatically elongating fromthe primers to generate an amplification product. Amplificationtypically requires the presence of deoxyribonucleoside triphosphates, aDNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate bufferand/or co-factors for optimal activity of the polymerase enzyme (e.g.,MgCl₂ and/or KCl).

The term “primer” is used herein as known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′—OH group whereto further“nucleotides” may be attached by a template-dependent DNA polymeraseestablishing 3′ to 5′ phosphodiester linkage whereby deoxynucleosidetriphosphates are used and whereby pyrophosphate is released. Therefore,there is—except possibly for the intended function—no fundamentaldifference between a “primer”, an “oligonucleotide”, or a “probe”.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet3:66-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 253389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., aC5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides are referredto herein or are otherwise known in the art. In certain embodiments,modified nucleotide substitutions modify melting temperatures (Tm) ofthe oligonucleotides relative to the melting temperatures ofcorresponding unmodified oligonucleotides. To further illustrate,certain modified nucleotide substitutions can reduce non-specificnucleic acid amplification (e.g., minimize primer dimer formation or thelike), increase the yield of an intended target amplicon, and/or thelike in some embodiments. Examples of these types of nucleic acidmodifications are described in, e.g., U.S. Pat. No. 6,001,611, which isincorporated herein by reference.

MTB-RIF and/or MTB-INH Nucleic Acids and Oligonucleotides

The present disclosure provides methods to detect MTB-RIF and/or MTB-INHby amplifying, for example, a portion of one or more of the rpoB, inhA,and katG nucleic acid sequences. Nucleic acid sequences for rpoB, inhA,and katG are available, e.g., through GenBank Specifically, primers andprobes to amplify and detect rpoB, inhA, and katG nucleic acid moleculetargets are provided by the embodiments in the present disclosure.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs: 1 through 409, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1through 409, or a complement of SEQ ID NOs: 1 through 409, and thevariant.

TABLE I  Probe for rpoB, inhA, and katG nucleic acid molecule targetsrpoB 531L TCG/TTG Ser/Leu SEQ Oligo Name ID NO: Sequence ModificationsRMRPO3SP531L09 1 FGTTGGQJGCTGGGGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2,J-G-clamp RMRPO3SP531L18 2 FACTGTTQGGLGLTGGGP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3SP531L19 3 FCTGTTQGGLGLUGGGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3S531L18B 4 FACTGTTQGGLGLUGGGP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S531L18C 5 FCTGTUQGGLGLUGGGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L1B 6 FCTGTTQGGLGCTGGGGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC RMRPO3SP531L20 7 FALUGTTQGGLGLUGGP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dU RMRPO3S531L20 8JCCGALTGTTGQGLGLUP J-Threo-JA270::P- Phosphate::Q-BHQ- 2, L =Propynyl dC, U = propynyl dU RMRPO3SP531L22 9 JCTGTTGGCGLUGQGGPJ-Threo-JA270::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3SP531L24 10 FLUGUUQGGLGLTGGGGLP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3SP531L25 11 FLUGUUQGGLGLTGGGGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3SP531L26 12 FUGUUGQGLGLTGGGGLLLP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S531L20B 13 FALUGUUQGGLGLUGGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3S531L20C 14 EALUGUUQGGLGLUGGP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S531L20D 15 EALUGUTQGGLGLUGGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3S531L25B 16 ELUGUUQGGLGLUGGGP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S531L25C 17 ELUGUUQGGLGLUGGLPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3S531L20F 18 EALUGUUQGGLGLUGLAGLP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3531L20HS 19 EALUGUUQJGGLGLUGGPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L25C2 20 ELUGUUGQGLGLUGGLP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3531L20C2 21 EALUGUUQGGLGLUGLPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L25C3  22 EALUGUUQGGLGLUGGLP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S531F1 23 EALUGUUQLGLGLUGGPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L25B2 24 ELUGUUQGGLGLTGGGP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3531L31 25 EUGUUGQGLGLTGGGGPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L25B3 26 ELUGUUGQGLGLTGGGP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3531L25B4 27 ELUGUUGQGLGLTGGCPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3F531L 28 FCCJACAQGTCGGCGCTTGP F-Threo-FAM::J-t-Butyl benzyl-dA::P-Phosphate::Q-BHQ-2 RMRPO3F531L02 29 FCCJACAQGTCGGCGCTTGTGGGTCPF-Threo-FAM::J-t-Butyl benzyl-dA::P- Phosphate::Q-BHQ-2 RMRPO3F531L04 30FCCAACAQGTLGGLGLTTGP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC RMRPO3F531L05 31 FCCAACQAGTLGGLGLTTGP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3F531L06 32FCCAACAQGULGGLGLTUGP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC, U = propynyl dU RMRPO3AP531L11 33 FCCAACAQGTJGGCGCTTGPF-Threo-FAM::P- Phosphate::Q-BHQ-2, J-G-clamp RMRPO3AP531L12 34FCCAAJAQGTCGGCGCTTGP F-Threo-FAM::P- Phosphate::Q-BHQ-2, J-G-clampRMRPO3AP531L13 35 FCCAACAQGTCGGJGCTTGP F-Threo-FAM::P-Phosphate::Q-BHQ-2, J-G-clamp RMRPO3AP531L14 36 FCCAACAQGTCGGCGJTTGPF-Threo-FAM::P- Phosphate::Q-BHQ-2, J-G-clamp RMRPO3A531L12B 37FCCAALQAGUCGGCGCTTGP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC, U = propynyl dU RMRPO3AP531L17 38 FCCAALAQGTLGGLGLPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3A531L17B 39FCCAALQAGTLGGLGLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dCRMRPO3A531L19 40 FCCAALQAGTLGGLGCTP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC RMRPO3A531L20 41 ECCAALAQGTLGGLGCTP E-Threo-HEX::P-Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3A531L18D 42JCCAALAGULGGCQGLTP J-Threo-JA270::P- Phosphate::Q-BHQ- 2, L =Propynyl dC, U = propynyl dU RMRPO3A531L12C 43 ECCAALQAGUCGGCGCTTGPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3A531L12D 44 ECCAALQAGULGGCGCTTGP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3531Ll7B2 45 ECCAALAQGULGGLGLPE-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3531L17B3 46 ELLAALAQGULGGLGLP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3A531L21 47 FLLAALQAGULGGLGLUPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3A531L22 48 FLLJALQAGULGGLGLP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU, J-G- clamp RMRPO3531L17B4 49FLLAALQAGULGGLGLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC,U = propynyl dU RMRPO3531L17B5 50 FLLJALQAGULGGLGLP E-Threo-HEX::P-Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dU,t-Butyl benzyl-dARMRPO3A531L20B 51 ELLAALAQGULGGLGLP E-Threo-HEX::P- Phosphate::Q-BHQ-2, L = Propynyl dC RM5L17B3a 52 ELLAALAQGTLGGLGLP E-Threo-HEX::P-Phosphate::Q-BHQ- 2, L = Propynyl dC rpoB 531W TCG/TGG Ser/Trp SEQOligo Name ID NO: Sequence Modifications RMRPO3F531W  53FCCLACAQGTCGGCGCTTGP F = Threo-FAM; Q = t- Butyl benzyl-dC; P =Phosphate; Q = BHQ-2 RMRPO3F531W02  54 FCCLACAQGTCGGCGCTTGTP F =Threo-FAM; L = t- Butyl benzyl-dC; P = Phosphate; Q = BHQ-2RMRPO3F531W06  55 FCCJACAQGTCGGCGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2,J-G-clamp rpoB 526L CAC/CTC His/Leu SEQ Oligo Name ID NO: SequenceModifications RMRPO3A07 56 FLUUGAGQGGULAALLLLP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dU RMRPO3A07B2 57FTTGAGGQGTLAALCCCGACGGGGP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC: U = propynyl dU RMRPO3A08 58 FALLLULQAAGLGLLGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dURMRPO3A08B 59 FALLLULQAAGLGLLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC: U = propynyl dU RMRPO3A08C 60 FALLLULQAAGCGLLGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dURMRPO3A09 61 JLUUGAGGGULAAQLLLLP J-JA270::P- Phosphate::Q-BHQ- 2, L =Propynyl dC: U = propynyl dU AYRPO3526LFM07 62 FLUUGAGGGQULAALLLLGAPF-Threo-FAM::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526LFM08 63FGLUUGAGQGGULAALLLLGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdUAYRPO3526LFM09 64 FGLUUGAGGQGULAALLLLGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526LFM10 65F GLUUGAGGGQULAALLLL GAP F-Threo-FAM::P- Phosphate::Q-BHQ-2::L-pdC::U-pdU AYRPO3526LFM11 66 FGLUUGAGGQGULAALLLLGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: J-G-clamp::L-pdC::  U-pdU AYRPO3526LFM12 67FGLUUGAGGGQUJAALLLLGAP F-Threo-FAM::P- Phosphate::Q-BHQ-24-G-clamp::L-pdC::  U-pdU AYRPO3526LJA01 68 FGLUUGAGGQGULAALLLLGAPF-th-JA270::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526LJA02 69FGLUUGAGGGQULAALLLLGAP F-th-JA270::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdUAYRPO3526LJA03 70 FGLUUGAGGQGJLAALLLLGAP F-th-JA270::P-Phosphate::Q-BHQ-2:: J-G-clamp::L-pdC::  U-pdU AYRPO3526LJA04 71FGLUUGAGGGQUJAALLLLGAP F-th-JA270::P- Phosphate::Q-BHQ-2::J-G-clamp::L-pdC::  U-pdU AYRPO3526LJA05 72 FGLUUGAGGGJQLAALLLLGAPF-th-JA270::P- Phosphate::Q-BHQ-2:: J-G-clamp::L-pdC::  U-pdUAYRPO3526LJA06 73 FGLUUGAGGGJLAAQLLLLGAP F-th-JA270::P-Phosphate::Q-BHQ-2:: G-clamp::L-pdC::  J-U-pdU rpoB 526Y CAC/TAC His/TyrSEQ Oligo Name ID NO: Sequence Modifications RMRPO3A06E 74JUUGUAGGULAALQLLLGAP J-JA270::P- Phosphate::Q-BHQ- 2, L = Propynyl dC,U = propynyl dU AYRPO3526YFM07 75 FLUUGUJGGQULAALLLLGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2::J- T-propdA::L-pdC::  U-pdU AYRPO3526YFM08 76FLUUGQUJGGULAALLLLGAP F-Threo-FAM::P- Phosphate::Q-BHQ-24-U-T-propdA:L-pdC::  pdU AYRPO3526YFM09 77 FLUUGUJGQGULAALLLLGAPF-Threo-FAM::P- Phosphate::Q-BHQ-2::J- T-propdA::L-pdC::  U-pdUAYRPO3526YFM10 78 FLUUGUAGQGULAALLLLGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526YHX01 79FLUUGUJGQGULAALLLLGAP F-Threo-HEX::P- Phosphate::Q-BHQ-2::J-T-propdA::L-pdC::  U-pdU AYRPO3526YHX02 80 FLUUGUAGQGULAALLLLGAPF-Threo-HEX::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526YHX03 81FUUGUJGQGULAALLLLGAP F-Threo-HEX::P- Phosphate::Q-BHQ-2::J-T-propdA::L-pdC::U- pdU AYRPO3526YHX04 82 FUUGUJGQGULAALLLLPF-Threo-HEX::P- Phosphate::Q-BHQ-2::J- T-propdA::L-pdC:: U-pdUAYRPO3526YHX05 83 FUUGUJQGGULAALLLLP F-Threo-HEX::P-Phosphate::Q-BHQ-2::J- T-propdA::L-pdC:: U-pdU AYRPO3526YHX06 84FLUUGUAGQGJLAALLLLGAP F-Threo-HEX::P- Phosphate::Q-BHQ-2::J-G-clamp::L-pdC::U- pdU AYRPO3526YHX07 85 FLUUGUAGQGULAALLLLGALAPF-Threo-HEX::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526YHX08 86FLGLUUGUAGQGULAALLLLGAP F-Threo-HEX::P- Phosphate::Q-BHQ-2::L-pdC::U-pdU AYRPO3526YHX09 87 FGLUUGUAGQGULAALLLLGALAP F-Threo-HEX::P-Phosphate::Q-BHQ-2:: L-pdC::U-pdU AYRPO3526YHX10 88FLGLUUGUAGQGULAALLLLGALAP  F-Threo-HEX::P- Phosphate::Q-BHQ-2::L-pdC::U-pdU rpoB: 526D CAC/GAC His/Asp SEQ Name ID NO SequenceModifications RMRPO3A03C 89 FLUUGULQGGUCAACLLLP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dU RMRPO3A03D 90JLUUGULGGULAAQLLCCP J-JA270::P- Phosphate::Q-BHQ- 2, L = Propynyl dC,U = propynyl dU AYRPO3526DFM03 91 FTGTJGQGTCAACCCCGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2::J- G-Clamp AYRPO3526DFM04 92 FTGTJGGQTCAACCCCGAPF-Threo-FAM::P- Phosphate::Q-BHQ-2::J- G-Clamp AYRPO3526DFM05 93FGTTGTQJGGTCAACCCCGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2::J- G-ClampAYRPO3526DFM06 94 FGTTGTJGGQTCAACCCCGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2::J- G-Clamp AYRPO3526DJA07 95 FUUGUJGGULAQALLLLPF-JA270::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU::J-G- Clamp AYRPO3526DJA0896 FLUUGUJGGULAQALLLLP F-JA270::P- Phosphate::Q-BHQ-2::L-pdC::U-pdU::J-G- Clamp AYRPO3526DJA09 97 FUUGUJGGULAQALLLLGAPF-JA270::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU::J-G- Clamp AYRPO3526DJA1098 FLUUGUJGGULAQALLLLGAP F-JA270::P- Phosphate::Q-BHQ-2::L-pdC::U-pdU::J-G- Clamp AYRPO3526DJA11 99 FGLUUGUJGGULAQALLLLGAPF-JA270::P- Phosphate::Q-BHQ-2:: L-pdC::U-pdU::J-G- Clamp rpoB 526NCAC/AAC His/Asn SEQ Oligo Name ID NO: Sequence ModificationsRMRPO3SP526R2 100 FACCAAQLAAGLGLLGP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC RMRPO3SP526R3 101 FALLAAQLAAGLGLLGP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3AP526R1 102FTTGTTQGGTLAALLCCGAP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC RMRPO3AP526N1 103 FUUGUUQGGULAALLLP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC RMRPO3AP526N2 104FUUGUUQGGULAALLLGP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dCRMRPO3AP526N2B 105 FUUGUUQGGULJJLLLP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC: U = propynyl dU RMRPO3SP526N4 106FALLAALQAAGLGLLGALUP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = PropynyldC: U = propynyl dU RMRPO3SP526N4B 107 EALLAALQAAGLGLLGALUPH-Threo-HEX::P- Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dURMRPO3SP526N4B2 108 EALLAAQLAAGLGLLGALP H-Threo-HEX::P-Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dU RMRPO3SP526N4B2b109  EALLAAQLAAGLGLLGAP H-Threo-HEX::P- Phosphate::Q-BHQ- 2, L =Propynyl dC: U = propynyl dU RMRPO3SP526N4B3 110 FLAALQAAGLGLLGALUGPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dURMRPO3SP526N4C 111 FALLAALQAAGLGLLGP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC RMRPO3SP526N5 112 FALLAAQLAAGLGLLGALUPF-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dURMRPO3AP526N6 113 FUUGUUQGGULAALLLLGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC: U = propynyl dU RMRPO3AP526N7 114FUUGUQUGGULAALLLLGAP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = PropynyldC: U = propynyl dU RMRPO3AP526N8 115 FUUGUUQGGULAALLLLP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dU RMRPO3AP526N9 116FUUGUUQGGULOOLLLLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = PropynyldC: U = propynyl dU, O-t-butyl benzyl dA RMRPO3AP526N1B  117 FUUGUUQGGULJJLLLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L = Propynyl dC,J-N6 methyl dA RMRPO3A526N8B 118 FUUGUUGQGULAALLLLP F-Threo-FAM::P-Phosphate::Q-BHQ- 2, L = Propynyl dC: U = propynyl dU rpoB 533 CTG/CCGLeu/Pro SEQ Oligo Name ID NO: Sequence Modifications RMRPO3H533P10B 119ECGCJGGQGGCCCGGCP E-Threo-HEX::P- Phosphate::Q-BHQ-2, J-G-clampRMRPO3H533P10D 120 FLGLLGGQGGLLLGGLGGP F-Threo-FAM::P-Phosphate::Q-BHQ-2, J-G-clamp, L = Propynyl dC RMRPO3H533P10 121ECGCCGGQGGCCCGGCGGP H-Threo-HEX::P- Phosphate::Q-BHQ-2 rpoB: 513LCAA/CTA Gln/Leu SEQ Name ID NO: Sequence Modifications JJS513L44PJ12 122FLAGLUGAGLLUAQUULP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS513L68PJ12 123 FLLUAUULAUGGAQLLAGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJS513L59PJ12 124 FLLAGLUGAGLLUQAUULPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJS513L101PJ12125 FAGLLUAUULAUGQGALLAGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS513L84PJ12 126 FGLLUAUULATGGQALLAGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L60PJ12 127 FUGAAUAGGLULAQGLUGPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513L68PJ12128 FLUGGULLAUGAAQTAGGP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p =phosphate JJA513L103PJ12 129 FUULUGGULLAUGQAAUAGGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L61PJ12 130 FAUGAAUAGGLULQAGLUPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513L76PJ12131 FUGAAUAGGLULAQGLUGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L59PJ12 132 FGAAUAGGLULAGQLUGGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L95PJ12 133FLAUGAAUAGGLUQLAGLUGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L924PJ2 134 FGAAUAGGLI_TLAGQUUGGLUP L = pdC, U =pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513L928PJ2 135FGAAUAGGUULAGQLUGGLUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L927PJ2 136 FGAAUAGGLUUAGQLUGGLUP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L9232PJ2 137FGAAUAGGLUGAGQLUGGLUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L9243PJ2 138 FGAAUAGGLULLGQLUGGLUP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L9217PJ2 139FGAAUAGGLULAGQLAGGLUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L9218PJ2 140 FGAAUAGGLULAGQAUGGLUP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L9222PJ2  141FGAAUAGGAULAGQLUGGLUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L75PJ12 142 FGAAUAGGLULAGQLUGGLP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513L75PCA12 143FGAAUAGGLULAGQLUGGLP L = pdC, U = pdU, F = CAL Fluor Red 635, Q =BHQ2, p = phosphate JJA513L75PCB12 144 FGAAUAGGLULAGQLUGGLP L = pdC, U =pdU, F = CAL Fluor Red 635 dT, Q = BHQ2, p = phosphate JJA513L77PJ12 145FAUGAAUAGGLULQAGLUGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513L93PJ12 146 FUGAAUAGGLULAQGLUGGLP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJS513L85PJ12 147FLLUAUULAUGGAQLLAGAP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate rpoB: 513K CAA/AAA Gln/Lys SEQ Name ID NO SequenceModifications JJS513K84PJ12 148 FAGLAAAUULAUGQGALLAP L = pdC, U =pdU, F = th- JA270, Q = BHQ2, p = phosphate JJS513K77PJ12 149FLLAGLUGAGLAAQAUULAP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS513K79PJ12 150 FAGLUGAGLAAAUQULAUGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJS513K85PJ12 151FGLAAAUULAUGGQALLAGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513K43PJ12 152 FAUUUGLULAGLUQGGLP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA513K77PJ12 153 FUGAAUUUGLULAQGLUGGPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513K60PJ12154 FGAAUUUGLULAGQLUGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513K76PJ12 155 FGAAUUUGLULAGQLUGGLP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513K76PCA12 156FGAAUUUGLULAGQLUGGLP L = pdC, U = pdU, F = CAL Fluor Red 635, Q =BHQ2, p = phosphate JJA513K76PCB12 157 FGAAUUUGLULAGQLUGGLP L = pdC, U =pdU, F = CAL Fluor Red 635 dT, Q = BHQ2, p = phosphate JJA513K44PJ12 158FAAUUUGLULAGLQUGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513K80PJ12 159 FLLAUGAAUUUGLQULAGLP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJA513K59PJ12 160 FAAUUUGLULAGLQUGGLPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513K75PJ12161 FAAUUUGLULAGQLUGGLUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate rpoB: 513P CAA/CCA Gln/Pro SEQ Name ID NO: SequenceModifications JJS513P66PJ12 162 FAGLLLAUULAUGQGALLP L = pdC, U =pdU, F = th- JA270, Q = BHQ2, p = phosphate JJS513P83PJ12 163FAGLLLAUULAUGQGALLAP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS513P68PJ12 164 FLLLAUULAUGGAQLLAGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JS513P81PJ12 165 FUGAGLLLAUULAQUGGALPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA513P60PJ12166 FUGAAUGGGLULAQGLUGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA513P76PJ12 167 FUGAAUGGGLTLAQGLUGGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJS513P66GJ12 168 FAGLLEAUULAUGQGALLPE = G-clamp, L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphateJJS513P83GJ12 169 FAGLLEAUULAUGQGALLAP E = G-clamp, L = pdC, U =pdU, F = th-JA270, Q = BHQ2, p = phosphate JJS513P84PJ12 170FGLLLAUULAUGGQALLAGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS513P82PJ12 171 FGAGLLLAUULAUQGGALLP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate JJS513P84GJ12 172FGLLEAUUCAUGGQALLAGP E = G-clamp, L = pdC, U = pdU, F = th-JA270, Q =BHQ2, p = phosphate JJS513P82GJ12 173 FGAGLLEAUULAUQGGALLP E =G-clamp, L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphaterpoB: 522L TCG/TTG Ser/Leu SEQ Name ID NO: Sequence ModificationsJJA522L50PJ12 174 FGGULAALLLLAAQLAGP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA522L18PJ12 175 FALLLLAALAGLGQGP L =pdC, U = pdU, F = th- JA270,Q = BHQ2, p = phosphate JJA522L16PJ12 176FLLLAALAGLGGGQUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA522L17PJ12 177 FLLLLAALAGLGGQGP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA522L83PJ12 178 FUGGGULAALLLLQAALAGPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522L32PJ12179 FAALLLLAALAGLQGGP L = pdC, I = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA522L33PJ12 180 FLAALLLLAALAGQLGP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA522L48PJ12 181 FULAALLLLAALAQGLGP L =pdC, U = pdU, F = th- JA270, Q = BHQ2,  p = phosphate JJA522L30PJ12 182FLLLLAALAGLGGQGUP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA522L31PJ12 183 FALLLLAALAGLGQGGP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA522L63PJ12 184 FULAALLLLAALAQGLGGP L =pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522L47PJ12 185FLAALLLLAALAGQLGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate rpoB: 522Q TCG/CAG Ser/Gln SEQ ID Name NO: SequenceModifications JJS522Q48J12 186 FCCGCTCGAGGGGQTTGAP F = th-JA270, Q =BHQ2, p = phosphate JJS522Q32J12 187 FCCCGCTGCAGGGQGTTP F =th-JA270, Q = BHQ2, p = phosphate JJS522Q49J12 188 FCGCTGCAGGGGTQTGACPF = th-JA270, Q = BHQ2, p = phosphate JJA522Q31J12 189FACCCCTGCAGCGQGGTP F = th-JA270, Q = BHQ2, p = phosphate JJA522Q33J12190 FCAACCCCTGCAGQCGGP F = th-JA270, Q = BHQ2, p = phosphateJJA522q43J12 191 FCCCTGCAGCGGGQTTGTP F = th-JA270, Q = BHQ2, p =phosphate JJA522Q31PJ12 192 FALLLLUGCAGLGQGGUP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJS522Q49PJ12 193 FLG LUGLAGGGGUQUGALPL = pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522Q18PJ12194 FALLLLUGLAGLLGQGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJS522Q34PJ12 195 FLGLYGLAGGGGUQUGAP L = pdC, U = pdU, F = th-JA270, Q = BHQ2, p = phosphate JJA522Q20PJ12 196 FLAALLLLUGLAGQLGP L =pdC, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522Q33PJ12 197FLAALLLLUGLAGQLGGP L = pdC, U = pdU, F = th- JA270, Q = BHQ2, p =phosphate JJA522Q47PJ12 198 FLAALLLLUGLAGQLGGGP L = pdC, U = pdU, F =th- JA270, Q = BHQ2, p = phosphate rpoB: 522W TCG/TGG Ser/Trp SEQ IDName NO: Sequence Modifications JJA522W33PJ12 199 FLAALLLLLALAGQLGP L =pdV, U = pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522W33GJ12 200FLAALLLLEALAGQLGP E = G-clamp, L = pdC, U = pdU, F = th-JA270, Q =BHQ2, p = phosphate JJA522W47J12 201 FLAALLLLLALAGQLGGP L = pdC, U =pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522W47GJ12 202FLAALLLLEALAGQLGGP E = G-clamp, L = pdC, U = pdU, F = th-JA270, Q =BHQ2, p = phosphate JJA522W48PJ12 203 FULAALLLLLALAQGLGP L = pdC, U =pdU, F = th- JA270, Q = BHQ2, p = phosphate JJA522W48GJ12 204FULAALLLLEALAQGLGP E = G-clamp, L = pdC, U = pdU, F = th-JA270, Q =BHQ2, p = phosphate rpoB 516V GAC/GTC Asp/Val RMRPO3F516V 205FCTGGJCQCATGAATTGGCTCP F-Threo-FAM::J-t-Butyl benzyl-dA::P-Phosphate::Q-BHQ-2 RMRPO3UNF516V  206 FCTGGACQCATGAATTGGCTCPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3SP516V 207FTGGTCQCAGAACAACCCGCTP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3SP516Y208 EATGTACQCAGAACAACCCGCTGP F-Threo-HEX::P- Phosphate::Q-BHQ-2RMRPO3AP516Y 209 ECTGGTACQATGAATTGGCTCP F-Threo-HEX::P-Phosphate::Q-BHQ-2 RMRPO3A2P516Y 210 ECTGGTACQATGAATTGGCTCAGCPF-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3F516V02 211FCTGGJCQLATGAATTGGLTLP F-Threo-FAM::P- Phosphate::Q-BHQ- 2, L =Propynyl dC,  J-t-Butyl benzyl-dA RMRPO3SP516V03 212FTGGTCQCAGAACAACCCGCTGCGGP  F-Threo-FAM::P- Phosphate::Q-BHQ-2RMRPO3SP516V04 213 FTGGTCQCAAAACAACCCGCTP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3SP516V05 214 FTGGTEQCAGAACAACCCGCTPF-Threo-FAM::P- Phosphate::Q-BHQ-2, E-G-clamp RMRPO3SP516V06 215FTGGTCQEAGAACAACCCGCTP F-Threo-FAM::P- Phosphate::Q-BHQ-2, E-G-clampRMRPO3SP516Y02 216 EATGTACQCAGAACAACCCGGGGTP E-Threo-HEX::P-Phosphate::Q-BHQ-2 RMRPO3SP516V07 217 FTGGTCQCAGAATAACCCGCTGCGGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3SP516V08 218FTGGTCQCAAAACAACCCGCTGCGGP F-Threo-FAM::P- Phosphate::Q-13HQ-2RMRPO3SP516V09 219 FGGTCCQAGAACAACCCGCTGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3SP516V10 220 FATGGTCQCAGAACAACCCGCTPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3SP516V11 221ECATGGTCQCAGAACAACCCGP H-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3SP516V12222 FATGGTCQCAGAACAACCGGTTGP F-Threo-FAM::P- Phosphate::Q-BHQ-2RMRPO3S516V11B 223 FCATGGTQCCAGAACAACCCGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3S516V11C 224 FLAUGGUQLLAGAALAALLLGPF-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dURMRPO3S516V11D 225 FCATGGQTCCAGAACAACCCGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3S516V11E 226 FATGGTQCCAGAACAACCCGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3S516V11F 227JCATGGTCCAGAAQCAACCCGP J-JA270::P- Phosphate::Q-BHQ-2 RMRPO3S516V11G 228JLAUGGULLAGAAQLAALP J-JA270::P- Phosphate::Q-BHQ-2 RMRPO3516V11C2 229FLAUGGUQLLJGAJLAJLP F-Threo-FAM::P- Phosphate::Q-BHQ-2, L = Propynyl dC,U = propynyl dU, J: N6-Benzyl dA RM516V11G2 230 FLLUGGUQLLAGAALAALPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3516V11G2 231 JLAUGGULLAGAAQLAPJ-JA270::P- Phosphate::Q-BHQ- 2, L = Propynyl dC, U = propynyl dURMRPO3516V11G3 232 JLAUGGTLLAGAAQLAP J-JA270::P- Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl dU RMRPO3S516V12B 233FCATGGTCQCAGAACAACCGGTTGP F-Threo-FAM::P- Phosphate::Q-BHQ-2RMRPO3516V11C4 234 FLAUGGUQLLAGAALAALLP F-Threo-FAM::P-Phosphate::Q-BHQ-2, L = Propynyl dC, U = propynyl du RMRPO3516V11E2 235FTGGTQCCAGAACAACCCGCTGP F-Threo-FAM::P- Phosphate::Q-BHQ-2RMRPO3516V11E3 236 FATGGTQCCAGAACAACAGTTGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3516V11E5 237 FTGGTQCCAGAACAACCCGCTPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3516V11E6 238FTGGTCCQAGAACAACCCGCTP F-Threo-FAM::P- Phosphate::Q-BHQ-2 rpoB 516YGAC/TAC Asp/Tyr SEQ Oligo Name ID NO: Sequence ModificationsRMRPO3SP516Y03 239 EATGTAJCQAGAACAACCCGCP E-Threo-HEX::P-Phosphate::Q-BHQ-2, J-G-clamp RMRPO3S516YB2 240 FATGTQACCAGAACAACCCGCTGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3SP516Y 241EATGTACQCAGAACAACCCGCTGP E-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3AP516Y242 ECTGGTACQATGAATTGGCTCP E-Threo-HEX::P- Phosphate::Q-BHQ-2RMRPO3A2P516Y 243 ECTGGTACQATGAATTGGCTCAGCP E-Threo-HEX::P-Phosphate::Q-BHQ-2 RMRPO3SP516Y02 244 EATGTACQCAGAACAACCCGGGGTPE-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3SP516Y4B 245EATGTACQCAGAACAACCCGCTGTP E-Threo-HEX::P- Phosphate::Q-BHQ-2RMRPO3S516YB 246 FATGTACQCAGAACAACCCGCTGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3S516YC 247 FATGTAQCCAGAACAACCCGCTGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 rpoB 533P CTG/CCG Leu/Pro SEQOligo Name ID NO: Sequence Modifications RMRPO3H533P10B 248ECGCJGGQGGCCCGGCP E-Threo-HEX::P- Phosphate::Q-BHQ-2, J-G-clampRMRPO3H533P10D 249 FLGLLGGQGGLLLGGLGGP F-Threo-FAM::P-Phosphate::Q-BHQ-2, J-G-clamp RMRPO3H533 250 ECCGGCGQCCGACAGTCGGCGPH-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P02 251ECCGGCGQCCGACAGTCGGP H-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P03252 ECCGGCGQCCTACAGTCGGCGP H-Threo-HEX::P- Phosphate::Q-BHQ-2RMRPO3H533P04 253 ECCGGCGQCCAACAGTCGGCGP H-Threo-HEX::P-Phosphate::Q-BHQ-2 RMRPO3H533P05 254 ECCGGCGQCCCACAGTCGGCGPH-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P06 255ECCGGCQACCGACAGTCGGP H-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P07256 ECCGGCGQTCGACAGTCGGP H-Threo-HEX::P- Phosphate::Q-BHQ-2RMRPO3H533P08 257 ECCGGCQGCCGACAGTCGGCP H-Threo-HEX::P-Phosphate::Q-BHQ-2 RMRPO3H533P09 258 ECCGGCQACCGACAGTCGGCPH-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P10 259 ECGCCGGQGGCCCGGCGGPH-Threo-HEX::P- Phosphate::Q-BH Q-2 RMRPO3H533P11 260 ECGCCGQGGGCCCGGCGPH-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P12 261 ECGCCGGQGGCCCGGCPH-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P8B 262ECCGGCQGCCGACAGTCGGP - H-Threo-HEX::P- Phosphate::Q-BHQ- 2 RMRPO3H533P8C263 ECCGGCQGCCGACAGTCGP H-Threo-HEX::P- Phosphate::Q-BHQ-2 RMRPO3H533P13264 FCGCCGQGGGCCGGCCP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3H533P10C265 FCGCCGGQGGCCCGGCGGP F-Threo-FAM:P- Phosphate::Q-BHQ-2 RMRPO3H533P1OE266 FCGCCGQGGGCCCGGCGGP F-Threo-FAM:P- Phosphate::Q-BHQ-2 RMRPO3533P10C2267 FCGCCGGQGGCCCGGCP F-Threo-FAM:P- Phosphate::Q-BHQ-2 RMRPO3533P12B268 FCGCCGQGGGCCCGGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3533P12C269 FCGCCQGGGGCCCGGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3533P12B2270 FCGCCGQGGGCCCGGCGCP F-Threo-FAM::P- Phosphate::Q-BHQ-2RMRPO3533P12C2 271 FCGCCQGGGGCCCGGCGGP F-Threo-FAM::P-Phosphate::Q-BHQ-2 RMRPO3533P12C3 272 FCGCCQGAGGCCCGGCGGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3533P12C4 273FAGCCQGGGGCCCGGCGGP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3533P13 274FCCGGGQGCCCGGCGGP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3533P14 275FCCGGGGQCCCGGCGGP F-Threo-FAM::P- Phosphate::Q-BHQ-2 RMRPO3H533P02B 276ECCGGCGQCCGACAGTCP H-Threo-HEX::P- Phosphate::Q-BHQ-2 SEQ rpoB 511PID NO: CTG/CCG Leu/Pro AYRPO3511PFM01 277 FCAGCQCGAGCCAATTCATGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 AYRPO3511PFM02 278FCAGCCQGAGCCAATTCATGP F-Threo-FAM::P- Phosphate::Q-BHQ-2 AYRPO3511PFM03279 FCAGCQJGAGCCAATTCATGP F-Threo-FAM::P- Phosphate::Q-BHQ-2:: J-G-ClampAYRPO3511PFM04 280 FCAGCJGQAGCCAATTCATGP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: J-G-Clamp AYRPO3511PFM05 281 FCAGCCGQAGCCAATTCATGPF-Threo-FAM::P- Phosphate::Q-BHQ-2 SEQ rpoB 526R ID NO: CAC/CGC His/ArgAYRPO3526RFM01 282 FCTTGCGQGGTCAACCCCGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2 AYRPO3526RFM02 283 FTGCGGGQTCAACCCCGAPF-Threo-FAM::P- Phosphate::Q-BHQ-2 AYRPO3526RFM03 284FTGCGGGTCQAACCCCGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2 AYRPO3526RFM04285 FCTTGCGGGQTCAACCCCGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2AYRPO3526RFM05 286 FCTTGJGQGGTCAACCCCGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: J-G-Clamp AYRPO3526RFM06 287 FTGJGGGQTCAACCCCGAPF-Threo-FAM::P- Phosphate::Q-BHQ-2:: J-G-Clamp AYRPO3526RFM07 288FTGJGGGTCQAACCCCGAP F-Threo-FAM::P- Phosphate::Q-BHQ-2:: J-G-ClampAYRPO3526RFM08 289 FCTTGJGGGQTCAACCCCGAP F-Threo-FAM::P-Phosphate::Q-BHQ-2:: J-G-Clamp katG 315I AGC/ATC SEQ Oligo Name ID NO:Sequence Modifications AYKAT315ICM01 290 FGATCACCATCGGCATCGAQF-Threo-Coum343::Q- BHQ-2 AYKAT315ICM02 291 FCACCATQCGGCATCGAGGTCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315ICM03 292FCATCGGQCATCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYKAT315ICM04 293 FCACCATQCGGCATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315ICM05 294 FATCGGCQATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315ICM06 295FAUCGGCQAUCGAGGUCGUAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYKAT3151CM07 296 FAULGGLQAULGAGGULGUAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2:: U-5-Propynyl du:: L-5-Propynyl dC AYKAT315ICM03a 297 FCATCGQGCATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315ICM03b 298FCATCGGCAQTCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYKAT315ICM03c 299 FCATCGGCATCQGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315ICM03d 300 FCATCGGCQATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315ICY03a1 301FCATCGQGCATCGAGGTCGTAP F-CY5.5::P- Phosphate::Q-BHQ-2 AYKAT315ICY03a2302 FCATCGGCAQTCGAGGTCGTAP F-CY5.5::P- Phosphate::Q-BHQ-2AYKAT315ICY03a3 303 FCATCGGCATCQGAGGTCGTAP F-CY5.5::P-Phosphate::Q-BHQ-2 AYKAT315ICY03a4 304 FCATCGGCATCGAQGGTCGTAPF-CY5.5::P- Phosphate::Q-BHQ-2 katG 315N AGC/AAC SEQ Oligo Name ID NO:Sequence Modifications AYKAT315NCM01 305 FCAACGQGCATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315NCM02 306FCAACGGCAQTCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BH Q-2AYKAT315NCM03 307 FCAACGGCATCQGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315NCM04 308 FCAACGGQCATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315NCM05 309FCAACGGCQATCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BH Q-2AYKAT315NCM04a  310 FCAACGGQCATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315NCM05a  311 FCAACGGCQATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315NCM07 312FCAALGQGCATLGAGGTLGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::L-5_Me_dC AYKAT315NCM08 313 FAACGGCQATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315NCM09 314 FCACCAAQCGGCATCGAGGTCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315NCM10 315FCACCAAQCGGCATCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYKAT315NCM11 316 FLAALGQGLATLGAGGTLGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315NCM12 317 FAALGGLQATLGAGGTLGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::L-5_Me_dC AYKAT315NCM13 318FLALLAAQLGGLATLGAGGTLP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::L-5_Me_dC AYKAT315NCM14 319 FLALLAAQLGGLATLGAGGTLGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2:L-5_Me_dC AYKAT315NCM15 320FLAALGQGLATLGAGGTLGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::L-5-Propynyl dC AYKAT315NCM16 321 FAALGGLQATLGAGGTLGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2:: L-5-Propynyl dC AYKAT315NCM17322 FLALLAAQLGGLATLGAGGTLP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::L-5-Propynyl dC AYKAT315NCM18 323 FLALLAAQLGGLATLGAGGTLGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2:: L-5-Propynyl dC AYKAT315NCM19324 FCAACQGGCATCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYKAT315NCM20 325 FCCAACGQGCATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYKAT315NCM21 326 FCCAACQGGCATCGAGGTCGTAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315NCM22 327FACCAACQGGCATCGAGGTCGTAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYKAT315NCM23 328 FACCAAQCGGCATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 katG 315T AGC/ACC SEQ Oligo Name ID NO: SequenceModifications AYKAT315TCM01 329 FCACCACQCGGCATCGAGGTCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYKAT315TCM02 330FCACCAJQCGGCATCGAGGTCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J-G-Clamp AYKAT315TCM03 331 FCAJCGGQCATCGAGGTCGTAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCM04 332 FCAJCGGQCATCGAGGTCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCM05 333FCACCAJQCGGCATCGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-ClampAYKAT315TCM05a 334 FCACCAQJCGGCATCGAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCM05b 335 FCACCAJCGQGCATCGAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCM05c 336FCACCAJCGGCQATCGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-ClampAYKAT315TCY05b1 337 FCACCAJCGQGCATCGAP F-CY5.5::P-Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCY05b2 338 FCACCAJCGGCQATCGAPF-CY5.5::P- Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315TCY05b3 339FCACCAJCGGCATQCGAP F-CY5.5::P- Phosphate::Q-BHQ-2::J- G-ClampAYKAT315TCY05b4 340 FCACCAJCGGCATCGAQ F-CY5.5::Q-BHQ-2::J- G-ClampkatG 315T2 AGC/ACA SEQ Oligo Name ID NO: Sequence ModificationsAYKAT315T2CM01 341 FCACCAQJAGGCATCGAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315T2CM02 342 FCACCAJAGQGCATCGAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-Clamp AYKAT315T2CM03 343FCACCAJAGGCQATCGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-ClampinhA Probe Designs inhA-15T C->T SEQ Oligo Name ID NO: SequenceModifications AYINHA15TCM01 344 FGCGAGAQTGATAGGTTGTCGGPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHA15TCM02 345FGAGATGQATAGGTTGTCGGGGTGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA15TCM03 346 FGLGAGAQUGAUAGGUUGULGGP F-Threo-Coum343::P-Phosphate::Q-BHQ- 2::L-5-Propynyl dC::U- 5-Propynyl dU AYINHA15TCM04 347FAGATGAQTAGGTTGTCGGGGTGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA15TCM05 348 FAGAUGAQUAGGUUGULGGGGUGAP F-Threo-Coum343::P-Phosphate::Q-BHQ- 2::L-5-Propynyl dC::U- 5-Propynyl dU AYINHA15TCM06 349FGATGATQAGGTTGTCGGGGTGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA15TCM04a 350 FAGATGATAQGGTTGTCGGGGTGAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYTNHA15TCM04b 351 FAGATGATAGGQTTGTCGGGGTGAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHA15TCM04c 352FAGATQGATAGGTTGTCGGGGTGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA15TCM04d 353 FAGATQGATAGGTTGTCGGGGTGAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHA15TCM07 354 FAGAUGAQUAGGUUGUCGGGGUGAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHA15TCM08355 FGAUGAQUAGGUUGUCGGGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYINHA15TCM09 356 FAUGAQUAGGUUGUCGGGGUGAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHA15TCM09a357 FAUGAUQAGGUUGUCGGGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYINHA15TCM09b 358 FATGAUQAGGUUGUCGGGGUGAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHA15TCM06a359 FGAUGAUQAGGUUGUCGGGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYINHA15TCM06b 360 FGATGAQUAGGUUGUCGGGGUGAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dUJFINHA15TCM06A_1 361 FGAUGAUQAGGUUGUCGJGGUGAP F-Threo-Coum343::P-Phosphate::Q-BHQ- 2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM06A_2 362FGAUGAUQAGGUUGUCGGJGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM06B_1 363FGATGAQUAGGUUGUCGJGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM06B_2 364FGATGAQUAGGUUGUCGGJGUGAP F-Threo- Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFYINHA15TCM09A_1 365FAUGAUQAGGUUGUCGJGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM09A_1 366FAUGAUQAGGUUGUCGGJGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM09B_1 367FATGAUQAGGUUGUCGJGGUGAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA15TCM09B_2 368FATGAUQAGGUUGUCGGJGUGAP F- Threo-Coum343 ::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG AYINHAR15TCM01 369 FCTATCAQTCTCGCCGCGGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHAR15TCM02 370FCTATCAQTCTCGCCGCGGCCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHAR15TCM03 371 FCTATCAQTCTC GCCGCGGCC GP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINFIAR15TCM04 372 FTATCATQCTCGCCGCGGCCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 inhA-8A T->A SEQ Oligo NameID NO: Sequence Modifications AYINHA8ACM01 373 FTAGGATQGTCGGGGTGACTGCCAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHA8ACM02 374FTAGGATQGTCGGGGTGACTGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA8ACM03 375 FGATAGGQATGTCGGGGTGACTGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHA8ACM04 376 FUAGGAUQGULGGGGUGALUPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHA8ACM05377 FTAGGAQTGTCGGGGTGACTGCCAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA8ACM06 378 FTAGGATGTQCGGGGTGACTGCCAP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHA8ACM07 379 FTAGGATGTCGQGGGTGACTGCCAPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHA8ACM08 380FTAGGQATGTCGGGGTGACTGCCAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHA8ACM08a 381 FUAGGQAUGUCGGGGUGACUGCCAP F-Threo-Coum343::P-Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHA8ACM08b 382FUAGGQAUGULGGGGUGALUGLLAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYINHAR8ACM01 383 FCGACATQCCTATCGTCTCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHAR8ACM02 384FGACATCQCTATCGTCTCGCCGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHAR8ACM03 385 FACATCCQTATCGTCTCGCCGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHAR8ACM04 386 FCATCCTQATCGTCTCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHAR8ACM05 387FCGACATQCCTATCGTCTCGCCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHAR8ACM02a 388 FGACATCQCTATCGTCTCGCCGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHAR8ACM02b 389 FGACATCCQTATCGTCTCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHAR8ACM02c 390FGACATCCTAQTCGTCTCGCCGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHAR8ACM02d 391 FGACATQCCTATCGTCTCGCCGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHAR8ACM02e 392 FGACATCQCUAUCGUCUCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHAR8ACM02f393 FGACATCCQUAUCGUCUCGCCGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU AYINHAR8ACM02g 394 FGACATCCUAQUCGUCUCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU AYINHAR8ACM02h395 FGACATQCCUAUCGUCUCGCCGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU JFINHA8ACM08A_1 396 FUAGGQAUGUCGJGGUGACUGCCAPF-Threo-Coum343::P- Phosphate::Q-BHQ- 2::U-5-Propynyl dU:: J-7_Dz_dGJFINHA8ACM08A_2 397 FUAGGQAUGUCGGJGUGACUGCCAP F-Threo-Coum343::P-Phosphate::Q-BHQ- 2::U-5-Propynyl dU:: J-7_Dz_dG JFINHA8ACM08B_1 398FUAGGQAUGULGJGGUGALUGLLAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU:: J-7_Dz_dG::L-5_Me_dC JFINHA8ACM08B_2 399FUAGGQAUGULGGJGUGALUGLLAP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::U-5-Propynyl dU: J-7_Dz_dG::L-5_Me_dC inhA-8C T->C SEQ Oligo NameID NO: Sequence Modifications AYINHA8ACM01 400 FTAGGCTQGTCGGGGTGACTGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHA8ACM02 401FTAGGJTQGTCGGGGTGACTGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J-G-Clamp AYINHA8ACM03 402 FGATAGGQJTGTCGGGGTGACTGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2::J- G-Clamp AYINHA8ACM04 403 FATAGGJQTGTCGGGGTGACTGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2::J- G-Clamp AYINHA8ACM05 404FAGGJTGQTCGGGGTGACTGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2::J-G-Clamp AYINHAR8CCM01 405 FCGACAGQCCTATCGTCTCGCCGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHAR8CCM02 406 FGACAGCQCTATCGTCTCGCCGCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2 AYINHAR8CCM03 407FACAGCCQTATCGTCTCGCCGCP F-Threo-Coum343::P- Phosphate::Q-BHQ-2AYINHAR8CCM04 408 FCAGCCTQATCGTCTCGCCGCP F-Threo-Coum343::P-Phosphate::Q-BHQ-2 AYINHAR8CCM05 409 FCGACAGQCCTATCGTCTCGCCPF-Threo-Coum343::P- Phosphate::Q-BHQ-2

In one embodiment, the above described a plurality of sets of rpoB,inhA, and katG primers and probes are used in order to provide fordetection of MTB-RIF and/or MTB-INH in a biological sample suspected ofcontaining MTB-RIF and/or MTB-INH. The sets of primers and probes maycomprise or consist of the primers and probes specific for the rpoB,inhA, and katG nucleic acid sequences, comprising or consisting of thenucleic acid sequences of SEQ ID NOs: 1 through 409. In anotherembodiment, the primers and probes for the rpoB, inhA, and katG targetscomprise or consist of a functionally active variant of any of theprimers of SEQ ID NOs: 1 through 409.

A functionally active variant of any of the probes of SEQ ID NOs: 1through 409 may be identified by using the probes in the disclosedmethod. A functionally active variant of a probe of any of the SEQ IDNOs: 1 through 409 pertains to a primer which provides a similar orhigher specificity and sensitivity in the method or kit described hereinas compared to the respective sequence of SEQ ID NOs: 1 through 409.

The variant may, e.g., vary from the sequence of SEQ ID NOs: 1 through409 by one or more nucleotide additions, deletions or substitutions suchas one or more nucleotide additions, deletions or substitutions at the5′ end and/or the 3′ end of the respective sequence of SEQ ID NOs: 1through 409. As detailed above, a primer (and/or probe) may bechemically modified, i.e., a primer and/or probe may comprise a modifiednucleotide or a non-nucleotide compound. A probe (or a primer) is then amodified oligonucleotide. “Modified nucleotides” (or “nucleotideanalogs”) differ from a natural “nucleotide” by some modification butstill consist of a base or base-like compound, a pentofuranosyl sugar ora pentofuranosyl sugar-like compound, a phosphate portion orphosphate-like portion, or combinations thereof. For example, a “label”may be attached to the base portion of a “nucleotide” whereby a“modified nucleotide” is obtained. A natural base in a “nucleotide” mayalso be replaced by, e.g., a 7-desazapurine whereby a “modifiednucleotide” is obtained as well. The terms “modified nucleotide” or“nucleotide analog” are used interchangeably in the present application.A “modified nucleoside” (or “nucleoside analog”) differs from a naturalnucleoside by some modification in the manner as outlined above for a“modified nucleotide” (or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the rpoB, inhA,and katG nucleic acid sequences, e.g., nucleic acids encodingalternative portions of rpoB, inhA, and katG can be designed using, forexample, a computer program such as OLIGO (Molecular Biology InsightsInc., Cascade, Colo.). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis), similar melting temperatures forthe members of a pair of primers, and the length of each primer (i.e.,the primers need to be long enough to anneal with sequence-specificityand to initiate synthesis but not so long that fidelity is reducedduring oligonucleotide synthesis). Typically, oligonucleotide primersare 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

In addition to a set of primers, the disclosed methods may use one ormore probes in order to detect the presence or absence of MTB-RIF and/orMTB-INH. The term “probe” refers to synthetically or biologicallyproduced nucleic acids (DNA or RNA), which by design or selection,contain specific nucleotide sequences that allow them to hybridize underdefined predetermined stringencies specifically (i.e., preferentially)to “target nucleic acids”, in the present case to a MTB-RIF and/orMTB-INH (target) nucleic acid. A “probe” can be referred to as a“detection probe” meaning that it detects the target nucleic acid.

In some embodiments, the described rpoB, inhA, and katG probes can belabeled with at least one fluorescent label. In one embodiment, therpoB, inhA, and katG probes can be labeled with a donor fluorescentmoiety, e.g., a fluorescent dye, and a corresponding acceptorfluorescent moiety, e.g., a quencher.

In one embodiment, the probes comprise or consist of a fluorescentmoiety and the nucleic acid sequences comprise or consist of SEQ ID NOs:1 through 409.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers. Embodiments mayuse a single probe or a pair of probes for detection of theamplification product. Depending on the embodiment, the probe(s) use maycomprise at least one label and/or at least one quencher moiety. As withthe primers, the probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18,20, 21, 22, 23, 24, or 25) nucleotides in length.

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described rpoB, inhA, and katG nucleic acid sequences. Aprimer can be purified from a restriction digest by conventionalmethods, or it can be produced synthetically. The primer is preferablysingle-stranded for maximum efficiency in amplification, but the primercan be double-stranded. Double-stranded primers are first denatured,i.e., treated to separate the strands. One method of denaturing doublestranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the described rpoB,inhA, and katG nucleic acid molecules. The temperature for annealing isusually from about 35° C. to about 65° C. (e.g., about 40° C. to about60° C.; about 45° C. to about 50° C.). Annealing times can be from about10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 secto about 40 sec). The reaction mixture is then adjusted to a temperatureat which the activity of the polymerase is promoted or optimized, i.e.,a temperature sufficient for extension to occur from the annealed primerto generate products complementary to the template nucleic acid. Thetemperature should be sufficient to synthesize an extension product fromeach primer that is annealed to a nucleic acid template, but should notbe so high as to denature an extension product from its complementarytemplate (e.g., the temperature for extension generally ranges fromabout 40° C. to about 80° C. (e.g., about 50° C. to about 70° C.; about60° C.). Extension times can be from about 10 sec to about 5 min (e.g.,about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30sec to about 2 min).

PCR assays can employ MTB-RIF and/or MTB-INH nucleic acid such as RNA orDNA (cDNA). The template nucleic acid need not be purified; it may be aminor fraction of a complex mixture, such as MTB-RIF and/or MTB-INHnucleic acid contained in human cells. MTB-RIF and/or MTB-INH nucleicacid molecules may be extracted from a biological sample by routinetechniques such as those described in Diagnostic Molecular Microbiology:Principles and Applications (Persing et al. (eds), 1993, AmericanSociety for Microbiology, Washington D.C.). Nucleic acids can beobtained from any number of sources, such as plasmids, or naturalsources including bacteria, yeast, viruses, organelles, or higherorganisms such as plants or animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KC, 10 mM Tris-HCl (pH 8.3),15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured template DNA, 50pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10%DMSO). The reactions usually contain 150 to 320 μM each of dATP, dCTP,dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target MTB-RIF and/or MTB-INH nucleic acid molecules. Thelimiting factors in the reaction are the amounts of primers,thermostable enzyme, and nucleoside triphosphates present in thereaction. The cycling steps (i.e., denaturation, annealing, andextension) are preferably repeated at least once. For use in detection,the number of cycling steps will depend, e.g., on the nature of thesample. If the sample is a complex mixture of nucleic acids, morecycling steps will be required to amplify the target sequence sufficientfor detection. Generally, the cycling steps are repeated at least about20 times, but may be repeated as many as 40, 60, or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength.

In one example, a oligonucleotide probe can contain a donor fluorescentmoiety and a corresponding quencher, which may or not be fluorescent,and which dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between thetwo fluorescent moieties such that fluorescent emission from the donorfluorescent moiety is quenched. During an extension step of a polymerasechain reaction, a probe bound to an amplification product is cleaved bythe 5′ to 3′ nuclease activity of, e.g., a Taq Polymerase such that thefluorescent emission of the donor fluorescent moiety is no longerquenched. Exemplary probes for this purpose are described in, e.g., U.S.Pat. Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly useddonor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchersare DABCYL and TAMRA. Commonly used dark quenchers include BlackHoleQuenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Calif.), IowaBlack™, (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry™Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the MTB-RIF and/or MTB-INH target nucleic acidsequence. Upon hybridization of the oligonucleotide probes to theamplification product nucleic acid at the appropriate positions, a FRETsignal is generated. Hybridization temperatures can range from about 35°C. to about 65° C. for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer, (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine×isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm is the distance in Angstroms (Å) from the nucleotide base tothe fluorescent moiety. In general, a linker arm is from about 10 Å toabout 25 Å. The linker arm may be of the kind described in WO 84/03285.WO 84/03285 also discloses methods for attaching linker arms to aparticular nucleotide base, and also for attaching fluorescent moietiesto a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide which contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of MTB-RIF and/or MTB-INH

The present disclosure provides methods for detecting the presence orabsence of MTB-RIF and/or MTB-INH in a biological or non-biologicalsample. Methods provided herein avoid problems of sample contamination,false negatives, and false positives. The methods include performing atleast one cycling step that includes amplifying a portion of rpoB, inhA,and katG target nucleic acid molecules from a sample using a pluralityof pairs of rpoB, inhA, and katG primers, and a FRET detecting step.Multiple cycling steps are performed, preferably in a thermocycler.Methods described herein can be performed using the rpoB, inhA, and katGprimers and probes to detect the presence of rpoB, inhA, and katGtargets, and the detection of the described SNPs in the rpoB, inhA, andkatG targets indicates the presence of MTB-RIF and/or MTB-INH in thesample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof the target nucleic acid. TaqMan® technology utilizes onesingle-stranded hybridization probe labeled with, e.g., one fluorescentdye and one quencher, which may or may not be fluorescent. When a firstfluorescent moiety is excited with light of a suitable wavelength, theabsorbed energy is transferred to a second fluorescent moiety accordingto the principles of FRET. The second fluorescent moiety is generally aquencher molecule. During the annealing step of the PCR reaction, thelabeled hybridization probe binds to the target DNA (i.e., theamplification product) and is degraded by the 5′ to 3′ nuclease activityof, e.g., the Taq Polymerase during the subsequent elongation phase. Asa result, the fluorescent moiety and the quencher moiety becomespatially separated from one another. As a consequence, upon excitationof the first fluorescent moiety in the absence of the quencher, thefluorescence emission from the first fluorescent moiety can be detected.By way of example, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems) uses TaqMan® technology, and is suitable for performing themethods described herein for detecting the presence or absence of thetarget nucleic acid in the sample.

Generally, the presence of FRET indicates the presence of MTB-RIF and/orMTB-INH in the sample, and the absence of FRET indicates the absence ofMTB-RIF and/or MTB-INH in the sample. Inadequate specimen collection,transportation delays, inappropriate transportation conditions, or useof certain collection swabs (calcium alginate or aluminum shaft) are allconditions that can affect the success and/or accuracy of a test result,however. Using the methods disclosed herein, detection of FRET within,e.g., 45 cycling steps is indicative of an MTB-RIF and/or MTB-INHinfection.

Representative biological samples that can be used include, but are notlimited to dermal swabs, nasal swabs, wound swabs, blood cultures, skin,and soft tissue infections. Collection and storage methods of biologicalsamples are known to those of skill in the art. Biological samples canbe processed (e.g., by nucleic acid extraction methods and/or kits knownin the art) to release MTB-RIF and/or MTB-INH nucleic acid or in somecases, the biological sample can be contacted directly with the PCRreaction components and the appropriate oligonucleotides.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can beused. In one embodiment, a LightCycler® instrument is used. Thefollowing patent applications describe real-time PCR as used in theLightCycler® technology. WO 97/46707, WO 97/46714, and WO 97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

It is understood that the embodiments described herein are not limitedby the configuration of one or more commercially available instruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect MTB-RIF and/or MTB-INH. An article ofmanufacture can include primers and probes used to detect rpoB, inhA,and katG, together with suitable packaging materials. Representativeprimers and probes for detection of MTB-RIF and/or MTB-INH are capableof hybridizing to rpoB, inhA, and katG target nucleic acid molecules. Inaddition, the kits may also include suitably packaged reagents andmaterials needed for DNA immobilization, hybridization, and detection,such solid supports, buffers, enzymes, and DNA standards. Methods ofdesigning primers and probes are disclosed herein, and representativeexamples of primers and probes that amplify and hybridize to rpoB, inhA,and katG target nucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling therpoB, inhA, and katG probes. Examples of suitable FRET donor fluorescentmoieties and corresponding acceptor fluorescent moieties are providedabove.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the rpoB, inhA, and katGprimers and probes to detect MTB-RIF and/or MTB-INH in a sample.Articles of manufacture may additionally include reagents for carryingout the methods disclosed herein (e.g., buffers, polymerase enzymes,co-factors, or agents to prevent contamination). Such reagents may bespecific for one of the commercially available instruments describedherein.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A method of detecting Mycobacterium tuberculosis(MTB) resistant to rifampicin (MTB-RIF) and/or MTB resistant toisoniazid (MTB-INH) in a sample, the method comprising a multiplexreal-time polymerase chain reaction assay comprising: (i) performing anamplifying step comprising contacting the sample with at least a set ofrpoB primers, a set of inhA primers, and a set of katG primers toproduce one or more amplification products if any rpoB, inhA, and katGtarget nucleic acid is present in the sample; (ii) performing ahybridizing step comprising contacting said one or more amplificationproducts with a plurality of detectable rpoB probes, a plurality ofdetectable inhA probes, and a plurality of detectable katG probescomprising: at least 17 detectable rpoB probes selected from the groupconsisting of SEQ ID NOs: 1 through 289, or a complement thereof, eachprobe having at least one label and optionally having at least onemodified nucleotide, and capable of detecting one or more of 17 singlenucleotide polymorphisms SNPs on rpoB which confer rifampicin resistanceto MTB, at least 3 detectable inhA probes selected from the groupconsisting of SEQ ID NOs: 344 through 409, or a complement thereof, eachprobe having at least one label and optionally having at least onemodified nucleotide, and capable of detecting one or more of 3 SNPs oninhA which confer isoniazid resistance to MTB, and at least 4 detectablekatG probes selected from the group consisting of SEQ ID NOs: 290through 343, or a complement thereof, each probe having at least onelabel, and optionally having at least one modified nucleotide andcapable of detecting one or more of 4 SNPs on katG which conferisoniazid resistance to MTB; wherein each of the plurality of detectablerpoB, inhA and katG probes do not detect wild-type MTB that is drugsensitive; and (iii) detecting the presence or absence of said one ormore amplification products, wherein the presence of said one or moreamplification products is indicative of the presence of MTB-RIF and/orMTB-INH in the sample and wherein the absence of said one or moreamplification products is indicative of the absence of MTB-RIF and/orMTB-INH in the sample; wherein said plurality of detectable rpoB probescomprise hydrolysis probes for detection of each one of the 17 SNPs onrpoB: rpoB 531L, rpoB 531W, rpoB 526L, rpoB 526Y, rpoB 526D, rpoB 526N,rpoB 513L, rpoB 513K, rpoB 513P, rpoB 522L, rpoB 522Q, rpoB 522W, rpoB516V, rpoB 516Y, rpoB 533P, rpoB 511P, and rpoB 526R; wherein saidplurality of detectable inhA probes comprise hydrolysis probes fordetection of each one of the 3 SNPs on inhA: inhA-15T, inhA-8A, andinhA-8C; and wherein said plurality of detectable katG probes comprisehydrolysis probes for detection of each one of the 4 SNPs on katG: katG3151, katG 315N, katG 315T, and katG 315T2.
 2. The method of claim 1,wherein: the plurality of detectable rpoB probes are selected from thegroup consisting of SEQ ID NOs: 52, 55, 58, 84, 99, 102, 108, 121, 142,155, 162, 180, 196, 199, 238, 240, 280, and 286, or a complementthereof; the plurality of detectable inhA probes are selected from thegroup consisting of SEQ ID NOs: 363, 384, and 407, or a complementthereof; and the plurality of detectable katG probes are selected fromthe group consisting of SEQ ID NOs: 297, 305, 335, and 341, or acomplement thereof.
 3. The method of claim 1, wherein each of theplurality of detectable rpoB, inhA, and katG probes is labeled with adonor fluorescent moiety and a corresponding acceptor fluorescentmoiety; and wherein the detecting step comprises detecting the presenceor absence of fluorescence resonance energy transfer (FRET) between thedonor fluorescent moiety and the acceptor fluorescent moiety of each ofthe rpoB, inhA, and katG probes, wherein the presence or absence offluorescence emission is indicative of the presence or absence ofMTB-RIF and/or MTB-INH in the sample.
 4. The method of claim 3, whereinsaid amplification employs a polymerase enzyme having 5′ to 3′ nucleaseactivity.
 5. The method of claim 3, wherein said acceptor fluorescentmoiety is a quencher.